The Serosal Immune System of the Thorax in Toxicology

The Serosal Immune System of the Thorax in Toxicology Abstract The thoracic cavities receive increasing attention in toxicology, because inhaled fibers and (nano)particles can reach these cavities and challenge the local lymphoid tissues. The thoracic and abdominopelvic cavities are controlled by the serosal immune system with its special, loosely organized lymphoid clusters, namely the fat-associated lymphoid clusters and milky spots, which together can be denoted as serosa-associated lymphoid clusters. These clusters house numerous innate lymphoid cells, namely the nonconventional, innate B lymphoid cell and innate lymphocyte type 2 populations. The fat depots in the thorax play a significant role in the serosal immunity, and they can be modulated by health issues such as metabolic syndrome. The serosal immune system operates in a unique way at the interface of the innate and acquired immunity and therefore exposure-related modulation of the system may have a distinct impact on the body’s immunity. To add to the investigation of the serosal immune system in the thorax, this review describes the (micro)anatomy of the immune system in relation to exposure, with a focus on the rat and mouse as preferred species in toxicology and immunology. fat-associated lymphoid clusters, innate lymphoid cells, mediastinum, milky spots, pleura, serosa-associated lymphoid clusters There is a growing interest in the abdominopelvic and thoracic cavities, because of health issues such as low-grade inflammation of their adipose tissue depots associated with obesity and metabolic syndrome. The thoracic adipose tissue depots in particular play an important role in cardiovascular disease and local immune responses (Cheung et al., 2013; Gómez-Hernandez et al. 2016). In toxicology, the thoracic cavities receive attention, because inhaled (nano)particles can be translocated from the lungs to the thoracic cavities and challenge its lymphoid tissues (Bénézech et al., 2015; Bernstein et al., 2015; Murphy et al., 2011). The immune system in mammals has anatomically distinct subsystems or compartments, each adapted to cope with antigens/pathogens present in a particular set of body organs and tissues (Janeway et al. 2001). At the same time all organ- and tissue-specific immune systems communicate and cooperate with each other and are served by the thymus and bone marrow; they act as “multiple sites, one system” (Harleman, 2000) or expressed otherwise, they act locally, but think systemically (Kuper et al., 2013). The mucosal immune system is currently addressed most. It is aimed to cope with the load of pathogens and antigens at the mucosal surfaces, while tolerating the microbiome and allowing food constituents to be taken up. The abdominopelvic and thoracic cavities are controlled by an organ- and tissue-specific immune system as well (Janeway et al., 2001), which has been named the celomic immune system after the shared embryonic origin of these cavities (Coelom-associated lymphomyeloid tissue, Lenzi et al., 1996; serous cavities of celomic origin, Ganshina, 2016), or the serosal immune system (after the serosal lining of the cavities and its associated lymphoid organs and tissues in analogy to the mucosal immune system). This review outlines the (micro)anatomic features of the serosal immune system in the thorax with a focus on the rat and mouse as preferred species in toxicology and immunology, and addresses its responses upon challenge. THE THORAX, BACKGROUND The thorax and abdomen in mammals develop from the celom during early embryogenesis. Eventually they are separated from each other by the diaphragm, and they are further subdivided in different cavities. In the thorax of rat and mouse, the 2 pleural cavities are left and right from the mediastinum (Figure 1A) and—in contrast to humans—there is an additional small cavity between the heart apex and the diaphragm (infracardiac space). Also in contrast to humans, the pleural cavities are not completely sealed off from each other. The actual spaces left in the cavities are rather small after being filled with the organs during embryonic development. Figure 1. View largeDownload slide A, Schematic presentation of the thorax (rat and mouse), divided into the 2 pleural cavities and the centrally located mediastinum containing the pericardial cavity and a small infracardiac cavity. The serosal membranes are 2-layered structures by folding back on itself (see the visceral- parietal and epicardial-pericardial transitions). *Adipose tissue depots. RPF = Retrocardiac pleural folds. (1) The parathymic or superior/cranial mediastinal LNs. (2) The left posterior/caudal mediastinal LN. (3) The right posterior or tracheobronchial LN. B, Schematic presentation of the rat pleural space. Immune cells and detached mesothelial cells float in the pleural fluid (Mouse: Total cells M: 5–10 × 105 and F: 15–20 × 105. Rat and mouse cell numbers based on bar charts by Scotland et al., 2011). M, males; F, females; MS, Milky spot; FALC, fat-associated lymphoid cluster. C, Schematic presentation of a drainage unit in the parietal pleura (based on rat, mouse and human data, peritoneum; rat data, pleura). The roof of the drainage unit with its stomata is called the cribriform membrane. Figure 1. View largeDownload slide A, Schematic presentation of the thorax (rat and mouse), divided into the 2 pleural cavities and the centrally located mediastinum containing the pericardial cavity and a small infracardiac cavity. The serosal membranes are 2-layered structures by folding back on itself (see the visceral- parietal and epicardial-pericardial transitions). *Adipose tissue depots. RPF = Retrocardiac pleural folds. (1) The parathymic or superior/cranial mediastinal LNs. (2) The left posterior/caudal mediastinal LN. (3) The right posterior or tracheobronchial LN. B, Schematic presentation of the rat pleural space. Immune cells and detached mesothelial cells float in the pleural fluid (Mouse: Total cells M: 5–10 × 105 and F: 15–20 × 105. Rat and mouse cell numbers based on bar charts by Scotland et al., 2011). M, males; F, females; MS, Milky spot; FALC, fat-associated lymphoid cluster. C, Schematic presentation of a drainage unit in the parietal pleura (based on rat, mouse and human data, peritoneum; rat data, pleura). The roof of the drainage unit with its stomata is called the cribriform membrane. The thoracic spaces and the diaphragm in mammals are lined by serous membranes (together denoted the serosa), which are named after their location (Figure 1A). The membranes are formed as a 2-layered structure by folding back on itself. The inner, visceral layer covers the organs; the outer, parietal layer covers the diaphragm and the ribcage (Figure 1B). In the rat and mouse, retrocardiac pleural folds extend from the diaphragm to the anterior end of the thoracic cavity, and from the pericardium to the sternum, lining the infracardiac space (Figure 1A; Cooray, 1949). In humans the heart rests directly onto the diaphragm and the 2 pleural folds are absent. The serosa consist of an outer mesothelial cell layer with basal lamina and 2–4 layers of submesothelial connective tissue with nerves, bloodvessels, and lymphatics. The thickness and histology varies between species and location (Agostoni and Zocchi, 2007). Rat and mouse have a thin visceral pleura. The thoracic adipose tissue depots are mostly restricted to the mediastinum (Figure 1A). These mediastinal fat depots can be quite extensive and contribute to metabolic syndrome (Cheung et al., 2013). The major mediastinal depot is at the hilus of the lungs. There is a smaller epicardial/pericoronary depot (Cheung et al., 2013; Fitzgibbons and Czech, 2014), and small ones in the right retrocardiac pleural fold and on the diaphragm in adult rats. The mediastinal fat in rat and mouse has mixed white and brown fat characteristics (“beige” or “brite”), also found in humans albeit that the majority of fat is white (De Jong et al., 2015; Fitzgibbons and Czech, 2014; Padilla et al., 2013). The thoracic spaces are filled with a slippery fluid to lubricate the organs and reduce the friction between organs and with the rib cage during movement. The fluid is reabsorbed by lymphatics and visceral pleural capillaries (Agostoni and Zocchi, 2007). Pleural and pericardial fluids contain free-floating immune cells and detached mesothelial cells (Figure 1B). The thoracic duct drains the right side of the thorax (Figure 1A) and the entire body below the diaphragm, and carries lymph and emulsified fats known as chyle. Leakage from the thoracic duct into the thorax is denoted chylothorax, which is milky-colored but watery in case of fasting. Stomata allow communication between the thoracic spaces and lymphatics (Figs. 1B and 1C). They appear unique to the parietal pleura and retrocardiac pleural folds (Wang et al., 1977). Stomata are present on the diaphragm of several species, albeit only or mainly at the peritoneal side (Bernstein et al., 2015). In rats, the presence of stomata at the pleural side might be restricted to the retrocardiac pleural folds which easily collapse onto the diaphragm. A group of stomata serve as a porte d’entree to a lacuna, together denoted the lymphatic drainage unit (Figure 1C). This unit leads to a lymph vessel (Li and Li, 2004). CAVITY-SPECIFIC LYMPHOID TISSUES: MILKY SPOTS AND FAT-ASSOCIATED LYMPHOID CLUSTERS GROUPED TOGETHER AS SEROSA-ASSOCIATED LYMPHOID CLUSTERS The serosal immune system of the thorax and abdominal cavities contains a special type of lymphoid tissues, termed milky spots (MSs) and (visceral) fat-associated lymphoid clusters (FALCs) (Figs. 2 and 3). MS have been distinguished from FALCs, mainly on the basis of location (MS: omentum vs FALCs: mesenteric, mediastinal gonadal, and pericardial fat) and on ontogeny (MS: before vs FALCs: after birth) (mainly mouse; Cruz-Migoni and Caamaño, 2016), but they probably cover identical structures albeit possibly with slightly different features depending on activation and perhaps location. Both MS and FALCs are close to stomata (rat; Wang et al., 2010). They share their association with the serosa and can therefore be denoted as serosa-associated lymphoid clusters (SALCs). Most information is based on SALCs in the peritoneal cavity; thoracic SALCs appear to be somewhat simpler in organization or less activated (mainly rat; pleura, pericardium; Michailova and Usunoff, 2006). Figure 2. View largeDownload slide Schematic presentation of a SALC in the mediastinum, mostly based on mouse data. HEV, high endothelial venule; NKT, natural killer cell; T = T lymphocyte; B1a and B1b, innate B lymphoid cells; B2, conventional B lymphocyte; ILC2, innate (T) lymphoid cell other than NKT cell; APC, antigen-presenting cell. Figure 2. View largeDownload slide Schematic presentation of a SALC in the mediastinum, mostly based on mouse data. HEV, high endothelial venule; NKT, natural killer cell; T = T lymphocyte; B1a and B1b, innate B lymphoid cells; B2, conventional B lymphocyte; ILC2, innate (T) lymphoid cell other than NKT cell; APC, antigen-presenting cell. Figure 3. View largeDownload slide Macrophage-rich and lymphocyte-rich SALCs, adult rats. A, Diaphragm with the transition between the broad muscular and the thin tendinous part (arrowhead) and the left retrocardiac pleural fold (arrow) with a few SALCs. B, Detail of a macrophage-rich SALC in the left pleural fold with numerous large macrophages, some lymphocytes, erythrocytes and a few granulocytes. C, A macrophage-rich SALC in the adipose tissue at the hilus of the lungs. D, Two lymphocyte-rich SALCs in the adipose tissue at the hilus of the lungs. E, Detail of 1 of the 2 SALCs from Figure 1D with an adipocyte and several bloodvessels. F, A small lymphocyte-rich SALC in brown adipose tissue on the heart. Hematoxylin & eosin (H&E)-stained sections. Figure 3. View largeDownload slide Macrophage-rich and lymphocyte-rich SALCs, adult rats. A, Diaphragm with the transition between the broad muscular and the thin tendinous part (arrowhead) and the left retrocardiac pleural fold (arrow) with a few SALCs. B, Detail of a macrophage-rich SALC in the left pleural fold with numerous large macrophages, some lymphocytes, erythrocytes and a few granulocytes. C, A macrophage-rich SALC in the adipose tissue at the hilus of the lungs. D, Two lymphocyte-rich SALCs in the adipose tissue at the hilus of the lungs. E, Detail of 1 of the 2 SALCs from Figure 1D with an adipocyte and several bloodvessels. F, A small lymphocyte-rich SALC in brown adipose tissue on the heart. Hematoxylin & eosin (H&E)-stained sections. In the thorax, the majority of lymphoid tissues and organs is located in the mediastinum (Figure 1A). The mediastinal pleura are considered to be the second site in the body with numerous MS/FALCs, after the omentum (mainly rat; Michailova and Usunoff, 2006; Wang et al., 2010), but in mice the pericardium may house even more SALCs (Bénézech et al., 2015). The number and size of SALCs in mediastinal fat can vary between strains of mice and were especially numerous in C57BL/6N (Elewa et al., 2014). SALCs are found also in the retrocardiac pleural folds of rat and mouse (Figure 3A) (Pereira de Sousa et al., 1994). In humans SALCs are located in the mediastinal pleura near the aorta, in the pleura over the esophagus and aorta and in the parietal pleural adipose tissue depots (Aharinejad et al., 1990). Ontogeny In mediastinal fat of mice, numbers of SALCs increase between 3- and 12 months-of-age (Elewa et al., 2014). In humans, pleural SALCs are completely developed at birth with multivacuolated adipocytes, lymphocytes and macrophages, and continue to develop until they reach maximum numbers in infancy (Aharinejad et al., 1990; Michailova and Usunoff, 2006). The numbers then start to decline. Based on omental SALCs in mice, the ontogeny is driven by other molecular events and cells than conventional secondary lymphoid organs, namely by tumor-necrosis factor (TNF) and type 2 innate lymphoid cells, and enhanced by invariant natural killer T cells (Cruz-Migoni and Caamaño, 2016; Perez-Shibayama and Ludewig, 2015). The chemokine Chemokine (C-X-C motif) ligand 13 (CXCL13) (sofar not detected in SALC of rats) is of great importance for the development of SALCs. Like Peyer’s patches, identifiable SALCs can increase in number upon an inflammatory challenge (mouse, omentum), via the recruitment of myeloid cells that express TNF necessary for signaling via the TNF receptors in stromal cells. The distinct postnatal increase in SALCs can lead to the impression that they develop essentially postnatally (Cruz-Mignoni and Caamaño, 2016; Jackson-Jones et al., 2016). Morphology SALCs are often described as aggregates of leukocytes around a central vessel or at a crossroad of vessels, although some authors also distinguish nonvascularized SALCs (Cruz-Migoni and Caamaño, 2016). The microvasculature of SALCs consists of a conventional succession of arteriole, precapillary, capillary, postcapillary, collecting venule, and venule. The capillaries may have dilated portions in the central zone of SALCs. The numbers of HEVs in mediastinal SALCs vary: Elewa et al. (2014) found PNAd+ plump endothelium in mediastinal SALCs of all 3 strains of mice investigated, but especially in the Th1-prone C57BL/6N strain with the largest and most numerous SALCs. There is convincing evidence that most mediastinal SALCs contain efferent lymphatics (lymphatic vessel endothelial receptor 1+ (LYVE-1+); Elewa et al., 2014). Afferent lymphatics appear absent and antigen and particles in the cavities may enter SALCs via the mesothelium, in the same way as (MALT) receives antigen via the specialized follicle-associated epithelium. The SALC-associated mesothelium is plump, and has many microvilli (mouse; mediastinum; Inoue and Otsuki, 1992). General features of SALCs are summarized in Table 1, and Figures 2 and 3. Distinct T and B compartmentalization appears absent. In mediastinal SALC in mice the majority of T cells was found at the base adjacent to adipocytes, while the B cells were located close to the mesothelium (mouse; mediastinum; Inoue and Otsuki, 1992), but other authors do not report this (Cruz-Migoni and Caamaño, 2016; Jaworska-Wilczynska et al., 2016). Iba1+ macrophages and Major histocompatibility complex (MHC) Class II+ antigen-presenting cells were distributed throughout the mediastinal SALCs of mice (Elewa et al., 2014). Germinal centers are absent or at least scarce (mouse; pericardium; Jackson-Jones et al., 2016). Follicular dendritic cells—the scaffold for germinal centers—have not been found in SALCs (mouse, thoracic para-aortic adipose tissue; Newland et al., 2017) and thus germinal center-like formations are probably simpler/more primitive than in MALT and LNs. Macrophage as well as lymphocyte-rich SALCs are present in the mediastinum (Figure 3). Table 1. Characteristics of SALCs, Compared with Conventional Secondary Lymphoid Organs, Tertiary Lymphoid Structures and “Crown-Like” Structures Characteristics  Spleen  LNs  Peyer’s Patches; NALT  Thorax SALCs  TLSa  CLSb  Encapsulation  √  √  —  —  —  —  HEVs  √  √  √  √  √  —  Afferent lymphatics  √  √  —  —  —  —  Efferent lymphatics  √  √  √  √  ?  —  Entrance of antigen or foreign material  Via afferent lymphatics  Via afferent lymphatics  Via follicular-associated epithelium, containing M cells  Via (specialized?) mesothelium  From surrounding, inflamed tissue  NA  T–B compartments  √  √  √  ?  ±  —  Germinal centres  √  √  √  Scarce, possibly simple/primitive follicles and germinal lefts, due to lack of scaffold (follicular dendritic cells)  √  —  Follicular dendritic cells  √  √  √  —  √  —  Innate T lymphoid ILC2 cells  Limited numbers  Scarce?  ?  Significant numbers, especially NHCs  ?  ?  Innate B1 B lymphoid cells  Limited numbers, mainly B1b B cells  —  Scarce?  Large numbers; the presence of B1a B cells in the rat is questionable  ?  ?  Characteristics  Spleen  LNs  Peyer’s Patches; NALT  Thorax SALCs  TLSa  CLSb  Encapsulation  √  √  —  —  —  —  HEVs  √  √  √  √  √  —  Afferent lymphatics  √  √  —  —  —  —  Efferent lymphatics  √  √  √  √  ?  —  Entrance of antigen or foreign material  Via afferent lymphatics  Via afferent lymphatics  Via follicular-associated epithelium, containing M cells  Via (specialized?) mesothelium  From surrounding, inflamed tissue  NA  T–B compartments  √  √  √  ?  ±  —  Germinal centres  √  √  √  Scarce, possibly simple/primitive follicles and germinal lefts, due to lack of scaffold (follicular dendritic cells)  √  —  Follicular dendritic cells  √  √  √  —  √  —  Innate T lymphoid ILC2 cells  Limited numbers  Scarce?  ?  Significant numbers, especially NHCs  ?  ?  Innate B1 B lymphoid cells  Limited numbers, mainly B1b B cells  —  Scarce?  Large numbers; the presence of B1a B cells in the rat is questionable  ?  ?  Abbreviations: NALT, nasopharynx-associated lymphoid tissue; TLS, tertiary lymphoid structures; CLS, Crown-like structures; NA, not applicable. a TLS, lymphocyte-specific microdomains in non-lymphoid organs, formed under chronic inflammatory conditions (Ager, 2017). b CLS, low-grade inflammation present in visceral fat of obese mice and humans with metabolic disease, but—unlike SALCs—also present in subcutaneous fat. Table 1. Characteristics of SALCs, Compared with Conventional Secondary Lymphoid Organs, Tertiary Lymphoid Structures and “Crown-Like” Structures Characteristics  Spleen  LNs  Peyer’s Patches; NALT  Thorax SALCs  TLSa  CLSb  Encapsulation  √  √  —  —  —  —  HEVs  √  √  √  √  √  —  Afferent lymphatics  √  √  —  —  —  —  Efferent lymphatics  √  √  √  √  ?  —  Entrance of antigen or foreign material  Via afferent lymphatics  Via afferent lymphatics  Via follicular-associated epithelium, containing M cells  Via (specialized?) mesothelium  From surrounding, inflamed tissue  NA  T–B compartments  √  √  √  ?  ±  —  Germinal centres  √  √  √  Scarce, possibly simple/primitive follicles and germinal lefts, due to lack of scaffold (follicular dendritic cells)  √  —  Follicular dendritic cells  √  √  √  —  √  —  Innate T lymphoid ILC2 cells  Limited numbers  Scarce?  ?  Significant numbers, especially NHCs  ?  ?  Innate B1 B lymphoid cells  Limited numbers, mainly B1b B cells  —  Scarce?  Large numbers; the presence of B1a B cells in the rat is questionable  ?  ?  Characteristics  Spleen  LNs  Peyer’s Patches; NALT  Thorax SALCs  TLSa  CLSb  Encapsulation  √  √  —  —  —  —  HEVs  √  √  √  √  √  —  Afferent lymphatics  √  √  —  —  —  —  Efferent lymphatics  √  √  √  √  ?  —  Entrance of antigen or foreign material  Via afferent lymphatics  Via afferent lymphatics  Via follicular-associated epithelium, containing M cells  Via (specialized?) mesothelium  From surrounding, inflamed tissue  NA  T–B compartments  √  √  √  ?  ±  —  Germinal centres  √  √  √  Scarce, possibly simple/primitive follicles and germinal lefts, due to lack of scaffold (follicular dendritic cells)  √  —  Follicular dendritic cells  √  √  √  —  √  —  Innate T lymphoid ILC2 cells  Limited numbers  Scarce?  ?  Significant numbers, especially NHCs  ?  ?  Innate B1 B lymphoid cells  Limited numbers, mainly B1b B cells  —  Scarce?  Large numbers; the presence of B1a B cells in the rat is questionable  ?  ?  Abbreviations: NALT, nasopharynx-associated lymphoid tissue; TLS, tertiary lymphoid structures; CLS, Crown-like structures; NA, not applicable. a TLS, lymphocyte-specific microdomains in non-lymphoid organs, formed under chronic inflammatory conditions (Ager, 2017). b CLS, low-grade inflammation present in visceral fat of obese mice and humans with metabolic disease, but—unlike SALCs—also present in subcutaneous fat. The mesothelial lining has an interrupted basal lamina, which facilitates macrophage and lymphocyte migration, and small groups of macrophages are located within the mesothelial lining. Thin, mainly unmyelinated nerve fibers innervate SALCs, in the vicinity of the mesothelial lining, close to the lymphatic vessels and between the cells (mainly rat, pleura, pericardium; Michailova and Usunoff, 2006). Lymphocyte Populations An important feature of SALCs in the abdominal and thoracic cavities is the presence of innate lymphoid cells in addition to the conventional lymphocyte subpopulations (mouse, Cruz-Mignoni and Caamaño, 2016; Elewa et al., 2014, 2016, 2017; Jackson-Jones et al., 2018; Koyasu and Moro, 2012; Saenz et al., 2010). SALCs contain innate lymphocyte type 2 (ILC2) cells (especially natural helper cells or NHCs), which can secrete type 2 cytokines (eg, interleukin-5, interleukin-13). About half of the B lymphocytes present in the pleural spaces are nonconventional, innate B lymphoid cell (B1) cells (sIgMhi, sIgDlow, CD11b+), which are nonconventional innate B lymphocytes (as opposed to the conventional Conventional B lymphocyte [B2] lymphocytes). B1 B cells are also present in the spleen and intestines, but they are CD11b−. CD5+ B1a cells, a subpopulation of B1 cells, are not well-documented in the rat and may be scarce (Berland and Wortis, 2002). Box 1 compares the properties of CD5+ innate B lymphoid cell (B1a), CD5– innate B lymphoid cell (B1b), and B2 cells, and their presence in rat, mouse, and man. Box 1. Comparison of B1 (Innate) and B2 (Conventional) Lymphocytes B1a and B1b cells develop early in the fetal liver and are self-renewing in situ; B2 cells develop late in the bone marrow and are replaced from the bone marrow. B1a and B1b cells are primarily located in the peritoneal and pleural cavities; B2 cells in the spleen, LNs and MALT. B1a cells have a high production of natural antibodies; production by B1b cells is poorly investigated; production by B2 cells is low. Antibody isotype of B1a and B1b cells is IgM and antibody avidity is low; isotypes of B2 are IgM, IgG, IgD, and IgA and their avidity is high B1a antigens are often carbohydrate-specific and rarely protein-specific; B1b antigens are possibly carbohydrate- and protein-specific; B2 antigens are protein-specific and rarely carbohydrate-specific. B1a memory development is little or nonexistent; B1b memory (IgM) and B2 memory is present. B1a responses are T cell-independent; B1b responses are largely T cell-independent; B2 responses are often T cell-dependent. Evidence for the presence of B1a cells is sufficient in mouse and man, but poor in rat; evidence for B1b cells is sufficient in mouse and rat, but limited in man; evidence for B2 cells is sufficient in mouse, rat and man (Cunningham et al., 2014; Perry et al., 2012; Popi et al., 2016) Function SALCs are secondary lymphoid organs, involved in the induction of adaptive immune responses, with antigen-presenting cells in the context of MHC II complexes and antigen-specific B cells undergoing expansion and Ig switching even in mice lacking spleen, lymph nodes (LNs), and Peyer’s patches (mouse; mediastinum and omentum; Jackson-Jones et al. 2016; Rangel-Moreno et al. 2009). However, Meza-Perez and Randall (2017; mouse; omentum) positioned them between secondary and tertiary lymphoid structures. SALCs are key in serous B cell homeostasis and activation, by retaining B1 cells in the serous cavities via high expression of the chemokine CXCL13 (mouse; Rangel-Moreno et al., 2009). The presence of B1 cells and ILC2 T cells strongly suggest that SALCs support and coordinate the activation of innate lymphocytes in early, acute immune responses (mouse; peritoneum; Bénézech et al., 2015). IMMUNE FUNCTIONS OF THE MESOTHELIUM AND VISCERAL ADIPOSE TISSUE Mesothelium The mesothelial cells are mostly flattened, but some are cuboidal or “cobble stone”-like (Jaworska-Wilczynska et al., 2016). The cuboidal mesothelial cells are often found lining SALCs; they have abundant microvilli and occasionally cilia and their cytoplasm is rich in organelles, including multilamellar bodies (Mutsaers et al., 2002). The multilamellar bodies resemble those of pneumocytes II, which produce lung surfactant. The mesothelium has many functions to maintain serosal homeostasis and repair (rat and mouse; pleura and omentum; Gupta and Gupta, 2015; Katz et al., 2011; Mutsaers et al., 2002). It is a player in innate and acquired immune mechanisms, namely as a nonprofessional antigen-presenting cell (expressing MHC class II) and as producer of cytokines, leukocyte chemoattractants and glycosaminoglycans. Adipose Tissue Apart from thermogenesis and energy storage and release, visceral adipocytes and their stromovasculature play a pivotal role in immunity (acquired immunity: Kaminski and Randall, 2010; innate immunity: Schaffler and Scholmerich, 2010). Most information is based on investigations of the white adipose tissue in the abdominal cavity, but apply also to the mixed white and brown adipose tissue in the thorax (Baragetti et al., 2016; Patil et al., 2014). The white adipocytes produce and secrete classical cytokines such as interleukin-6 and TNF, and hormones like leptin. Leptin regulates macrophage function and in a broader sense modulates the immune functions of adipocytes by eg, inducing Toll-like receptor expression (Schaffler and Scholmerich, 2010). Adipocytes express multiple Toll-like receptors to recognize pathogens and to initiate immune responses, and they can secrete a variety of monocyte/macrophage chemoattractant molecules (Schaffler and Scholmerich, 2010; Dalmas et al., 2011). Interestingly, the innate immune system itself influences beige fat formation, via type 2 cytokine signaling (Kissig et al., 2016). For example, interleukin-4 production in white adipose tissue stimulates macrophages to produce norepinephrine, which leads to beige fat activation. The immune system-related aspects of the adipose tissue may depend on the location (Pond, 2005): adipocytes around LNs and omental SALCs are relatively small compared with those further away, respond more strongly to cytokines, and neighbor numerous antigen-presenting dendritic cells. Moreover, adipose tissue around the inguinal LN of mice contained less gamma/delta T lymphocytes and NK cells than epidydimal fat (Caspar-Bauguil et al., 2005). In the rat, perivascular adipose tissue in the abdomen express more inflammatory genes and markers of immune cells than the perivascular fat in the thorax, possibly because the abdominal adipose tissue has white adipose tissue characteristics, whereas in the thorax it is beige (Padilla et al., 2013). SALCs AND THORACIC FLUID RESPONSES TO EXPOSURE AND IN DISEASE The responses of mediastinal SALCs and thoracic fluid to inflammogenic stimuli, mostly inhaled or instilled particles, are summarized in Box 2 (rat and mouse; Bernstein et al., 2015; Broaddus et al., 2011; McKenzie and Caamaño, 2015; Mercer et al., 2013; Murphy et al., 2011; Ryman-Rasmussen et al., 2009; Gelzleichter et al., 1999; Choe et al., 1997; Peao et al., 1992; Bénézech et al., 2015; Jackson-Jones et al., 2016; Cooray, 1949; Pereira de Sousa et al., 1994). Acceleration of fluid flow happens by widening of already opened stomata and opening of formerly closed stomata, by increased drainage of the pleural interstitium and by increased diffusion and transcytosis (Bodega and Agostoni, 2004; Li and Li, 2004). Box 1. Exposure-Induced Changes in Mesothelium, Thoracic Fluid and Mediastinal SALCs (MS and FALCs) of Rodents Flat mesothelial cells become cuboidal and release inflammatory mediators (mouse; thoracic serosa); mesothelial cells may proliferate (rat, mouse; pleura). The fluid flow from the cavities into the lymphatics accelerates (rat; pleural space), and harbor increased numbers of inflammatory cells (rat; pleural space). Macrophages are recruited and activated, comparable to alveolar macrophages (rat; pleura, pleural space). Local serosal inflammation may occur (rat, mouse; pleura). SALCs increase in size and cellularity, and may fuse (rat, mouse; mediastinum); they may also develop germinal center-like B2 cells (GL7 + Ki67+) and germinal center-like structures (mouse; mediastinum). Particles can accumulate in SALCs and may induce granulomata and cell death in SALCs (rat; mediastinum). In humans, activation of SALCs has been linked also to inhalation of particles (Boutin et al., 1996; Mitchev et al., 2002). Black spots on the parietal pleura can be anthracotic SALCs, whereas hyaline pleural plaques, associated with exposure to asbestos, may be anthracotic SALCs or inflammatory processes on the parietal pleura as a result of blockage of the stomata by fibers (Murphy et al., 2011). Based on the stimulus, either mainly thoracic or abdominal SALCs react. For example omental SALCs reacted much more than pleural SALCs upon percutaneous injection with Schistosoma mansonii in mice, a nematode with preference to the mesenteric veins (Panasco et al., 2010). The opposite happened upon a subcutaneous injection with Litomosoides sigmodontis, a nematode that resides in the pleural cavity (Jackson-Jones et al., 2016). Particles and particle-containing phagocytes are thought to be removed from the pleural space via the stomata and associated lymphatic channels, but Lehnert (1992) questioned this route as the only or most important pathway. Based mainly on rat and some mouse and human data, he hypothesized that the caudal mediastinal tissue acts as the primary site via which particle-containing phagocytes are removed from the pleural space, to end up in the local LNs. Diseases can also affect SALCs. The mediastinal SALCs in the autoimmune MRL/MpJ-lpr mouse (prone to a systemic lupus erythematosus-like disease) and the BXSB/MpJ-Yaa mouse (prone to systemic autoimmunity) were much larger, with high numbers of T and B cells, macrophages and proliferating cells than their respective controls (Elewa et al., 2016). The size of SALCs correlated well with immune cell infiltration in the lungs. During pleural and lung inflammation, pleural B1 cells migrate to SALCs of the mediastinum and pericardium and produce IgM in the thoracic cavities (Jackson-Jones and Benezech, 2018). Moreover, B1 cells in peri-aortic SALCs appear to protect against (local) atherosclerosis (Srikakulapu et al., 2017). Pleural fluid cell numbers are increased in humans and mice with systemic autoimmune diseases (Pfau et al., 2014). SALCs may play a role in cancer metastasis as well, as shown in the peritoneum: some cancer cells expressed the receptors chemokine (C-C) receptor 4 and chemokine (C-X-C) receptor 4, which can favor their migration to SALCs, because their cell populations can express their ligands (Chemokine (C-C) motif 22[CCL22] and CXCL12) (Cruz-Migoni and Caamaño, 2016). DISCUSSION AND FUTURE DIRECTIONS The serosal immune system has unique anatomic and morphologic features, namely the lymphoid structures (SALCs), the large quantity of innate B1 B and ILC2 lymphoid cells and the lymphatic drainage units. The immune physiology of the serosal cavities is still poorly understood and many questions still remain. Do the abdominal and thoracic cavities co-operate like the respiratory tract and gut in the mucosal immune system? The different cavities start as 1 celom in embryonic life, but after birth there is no bulk flow between the pleural and peritoneal cavities (Grimaldi et al., 2006). Still, ip-injected material in the rat is rapidly transported through lymphatics to the LNs in the mediastinum (Shibata et al., 2007). This suggests a co-operation between the cavities on both sides of the diaphragm, albeit possibly only under “overload” conditions. Moreover, experiments with nematode-infected mice suggest that the cavities act largely as independent environments (Panasco et al., 2010). Another question is whether the mesothelial epithelium lining the SALCs can take up antigens and act in a way comparable to the follicle-associated epithelium of Peyer’s patches and nasopharynx-associated lymphoid tissue? Interestingly in animals with an undivided celom, like for example insects, the serosal epithelium is immune-competent and expresses many genes involved in bacterial recognition and transduction of this recognition to receptor activation (Jacobs et al., 2014). As such, it is especially involved in acute and mainly innate immune responses. B1 cells in the cavities not only protect local tissues, but also the mucosae with an acute innate immune response (Cruz-Migoni and Caamaño, 2016; Jackson-Jones et al., 2016; Symowski and Voehringer, 2017). Thus, the serosal immune system operates in a unique way at the interface of the innate and acquired immunity. Therefore exposure-related modulation of this system may have an enormous impact on the body’s immunity. In addition, evaluation of potential toxicity to SALCs can increase our understanding of the normal physiology of these clusters. Knowledge of SALCs locations, the variation in their morphology and their position in the serosal immune system is a first step to examine their response to exposure. ACKNOWLEDGMENTS The authors would like to thank Fariza Bouallala for her enthusiastic and valuable efforts at the early stage of the project and Darryl Leydekkers, Lisanne Meijer, Tim van Olmen, and Eva Rennen for their expert technical contribution. REFERENCES Ager A. ( 2017). High endothelial venules and other blood vessels: Critical organizers in lymphoid organ development and function. Front. Immunol . 8, 45. Google Scholar CrossRef Search ADS   Agostoni E., Zocchi L. ( 2007). 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The Serosal Immune System of the Thorax in Toxicology

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Abstract

Abstract The thoracic cavities receive increasing attention in toxicology, because inhaled fibers and (nano)particles can reach these cavities and challenge the local lymphoid tissues. The thoracic and abdominopelvic cavities are controlled by the serosal immune system with its special, loosely organized lymphoid clusters, namely the fat-associated lymphoid clusters and milky spots, which together can be denoted as serosa-associated lymphoid clusters. These clusters house numerous innate lymphoid cells, namely the nonconventional, innate B lymphoid cell and innate lymphocyte type 2 populations. The fat depots in the thorax play a significant role in the serosal immunity, and they can be modulated by health issues such as metabolic syndrome. The serosal immune system operates in a unique way at the interface of the innate and acquired immunity and therefore exposure-related modulation of the system may have a distinct impact on the body’s immunity. To add to the investigation of the serosal immune system in the thorax, this review describes the (micro)anatomy of the immune system in relation to exposure, with a focus on the rat and mouse as preferred species in toxicology and immunology. fat-associated lymphoid clusters, innate lymphoid cells, mediastinum, milky spots, pleura, serosa-associated lymphoid clusters There is a growing interest in the abdominopelvic and thoracic cavities, because of health issues such as low-grade inflammation of their adipose tissue depots associated with obesity and metabolic syndrome. The thoracic adipose tissue depots in particular play an important role in cardiovascular disease and local immune responses (Cheung et al., 2013; Gómez-Hernandez et al. 2016). In toxicology, the thoracic cavities receive attention, because inhaled (nano)particles can be translocated from the lungs to the thoracic cavities and challenge its lymphoid tissues (Bénézech et al., 2015; Bernstein et al., 2015; Murphy et al., 2011). The immune system in mammals has anatomically distinct subsystems or compartments, each adapted to cope with antigens/pathogens present in a particular set of body organs and tissues (Janeway et al. 2001). At the same time all organ- and tissue-specific immune systems communicate and cooperate with each other and are served by the thymus and bone marrow; they act as “multiple sites, one system” (Harleman, 2000) or expressed otherwise, they act locally, but think systemically (Kuper et al., 2013). The mucosal immune system is currently addressed most. It is aimed to cope with the load of pathogens and antigens at the mucosal surfaces, while tolerating the microbiome and allowing food constituents to be taken up. The abdominopelvic and thoracic cavities are controlled by an organ- and tissue-specific immune system as well (Janeway et al., 2001), which has been named the celomic immune system after the shared embryonic origin of these cavities (Coelom-associated lymphomyeloid tissue, Lenzi et al., 1996; serous cavities of celomic origin, Ganshina, 2016), or the serosal immune system (after the serosal lining of the cavities and its associated lymphoid organs and tissues in analogy to the mucosal immune system). This review outlines the (micro)anatomic features of the serosal immune system in the thorax with a focus on the rat and mouse as preferred species in toxicology and immunology, and addresses its responses upon challenge. THE THORAX, BACKGROUND The thorax and abdomen in mammals develop from the celom during early embryogenesis. Eventually they are separated from each other by the diaphragm, and they are further subdivided in different cavities. In the thorax of rat and mouse, the 2 pleural cavities are left and right from the mediastinum (Figure 1A) and—in contrast to humans—there is an additional small cavity between the heart apex and the diaphragm (infracardiac space). Also in contrast to humans, the pleural cavities are not completely sealed off from each other. The actual spaces left in the cavities are rather small after being filled with the organs during embryonic development. Figure 1. View largeDownload slide A, Schematic presentation of the thorax (rat and mouse), divided into the 2 pleural cavities and the centrally located mediastinum containing the pericardial cavity and a small infracardiac cavity. The serosal membranes are 2-layered structures by folding back on itself (see the visceral- parietal and epicardial-pericardial transitions). *Adipose tissue depots. RPF = Retrocardiac pleural folds. (1) The parathymic or superior/cranial mediastinal LNs. (2) The left posterior/caudal mediastinal LN. (3) The right posterior or tracheobronchial LN. B, Schematic presentation of the rat pleural space. Immune cells and detached mesothelial cells float in the pleural fluid (Mouse: Total cells M: 5–10 × 105 and F: 15–20 × 105. Rat and mouse cell numbers based on bar charts by Scotland et al., 2011). M, males; F, females; MS, Milky spot; FALC, fat-associated lymphoid cluster. C, Schematic presentation of a drainage unit in the parietal pleura (based on rat, mouse and human data, peritoneum; rat data, pleura). The roof of the drainage unit with its stomata is called the cribriform membrane. Figure 1. View largeDownload slide A, Schematic presentation of the thorax (rat and mouse), divided into the 2 pleural cavities and the centrally located mediastinum containing the pericardial cavity and a small infracardiac cavity. The serosal membranes are 2-layered structures by folding back on itself (see the visceral- parietal and epicardial-pericardial transitions). *Adipose tissue depots. RPF = Retrocardiac pleural folds. (1) The parathymic or superior/cranial mediastinal LNs. (2) The left posterior/caudal mediastinal LN. (3) The right posterior or tracheobronchial LN. B, Schematic presentation of the rat pleural space. Immune cells and detached mesothelial cells float in the pleural fluid (Mouse: Total cells M: 5–10 × 105 and F: 15–20 × 105. Rat and mouse cell numbers based on bar charts by Scotland et al., 2011). M, males; F, females; MS, Milky spot; FALC, fat-associated lymphoid cluster. C, Schematic presentation of a drainage unit in the parietal pleura (based on rat, mouse and human data, peritoneum; rat data, pleura). The roof of the drainage unit with its stomata is called the cribriform membrane. The thoracic spaces and the diaphragm in mammals are lined by serous membranes (together denoted the serosa), which are named after their location (Figure 1A). The membranes are formed as a 2-layered structure by folding back on itself. The inner, visceral layer covers the organs; the outer, parietal layer covers the diaphragm and the ribcage (Figure 1B). In the rat and mouse, retrocardiac pleural folds extend from the diaphragm to the anterior end of the thoracic cavity, and from the pericardium to the sternum, lining the infracardiac space (Figure 1A; Cooray, 1949). In humans the heart rests directly onto the diaphragm and the 2 pleural folds are absent. The serosa consist of an outer mesothelial cell layer with basal lamina and 2–4 layers of submesothelial connective tissue with nerves, bloodvessels, and lymphatics. The thickness and histology varies between species and location (Agostoni and Zocchi, 2007). Rat and mouse have a thin visceral pleura. The thoracic adipose tissue depots are mostly restricted to the mediastinum (Figure 1A). These mediastinal fat depots can be quite extensive and contribute to metabolic syndrome (Cheung et al., 2013). The major mediastinal depot is at the hilus of the lungs. There is a smaller epicardial/pericoronary depot (Cheung et al., 2013; Fitzgibbons and Czech, 2014), and small ones in the right retrocardiac pleural fold and on the diaphragm in adult rats. The mediastinal fat in rat and mouse has mixed white and brown fat characteristics (“beige” or “brite”), also found in humans albeit that the majority of fat is white (De Jong et al., 2015; Fitzgibbons and Czech, 2014; Padilla et al., 2013). The thoracic spaces are filled with a slippery fluid to lubricate the organs and reduce the friction between organs and with the rib cage during movement. The fluid is reabsorbed by lymphatics and visceral pleural capillaries (Agostoni and Zocchi, 2007). Pleural and pericardial fluids contain free-floating immune cells and detached mesothelial cells (Figure 1B). The thoracic duct drains the right side of the thorax (Figure 1A) and the entire body below the diaphragm, and carries lymph and emulsified fats known as chyle. Leakage from the thoracic duct into the thorax is denoted chylothorax, which is milky-colored but watery in case of fasting. Stomata allow communication between the thoracic spaces and lymphatics (Figs. 1B and 1C). They appear unique to the parietal pleura and retrocardiac pleural folds (Wang et al., 1977). Stomata are present on the diaphragm of several species, albeit only or mainly at the peritoneal side (Bernstein et al., 2015). In rats, the presence of stomata at the pleural side might be restricted to the retrocardiac pleural folds which easily collapse onto the diaphragm. A group of stomata serve as a porte d’entree to a lacuna, together denoted the lymphatic drainage unit (Figure 1C). This unit leads to a lymph vessel (Li and Li, 2004). CAVITY-SPECIFIC LYMPHOID TISSUES: MILKY SPOTS AND FAT-ASSOCIATED LYMPHOID CLUSTERS GROUPED TOGETHER AS SEROSA-ASSOCIATED LYMPHOID CLUSTERS The serosal immune system of the thorax and abdominal cavities contains a special type of lymphoid tissues, termed milky spots (MSs) and (visceral) fat-associated lymphoid clusters (FALCs) (Figs. 2 and 3). MS have been distinguished from FALCs, mainly on the basis of location (MS: omentum vs FALCs: mesenteric, mediastinal gonadal, and pericardial fat) and on ontogeny (MS: before vs FALCs: after birth) (mainly mouse; Cruz-Migoni and Caamaño, 2016), but they probably cover identical structures albeit possibly with slightly different features depending on activation and perhaps location. Both MS and FALCs are close to stomata (rat; Wang et al., 2010). They share their association with the serosa and can therefore be denoted as serosa-associated lymphoid clusters (SALCs). Most information is based on SALCs in the peritoneal cavity; thoracic SALCs appear to be somewhat simpler in organization or less activated (mainly rat; pleura, pericardium; Michailova and Usunoff, 2006). Figure 2. View largeDownload slide Schematic presentation of a SALC in the mediastinum, mostly based on mouse data. HEV, high endothelial venule; NKT, natural killer cell; T = T lymphocyte; B1a and B1b, innate B lymphoid cells; B2, conventional B lymphocyte; ILC2, innate (T) lymphoid cell other than NKT cell; APC, antigen-presenting cell. Figure 2. View largeDownload slide Schematic presentation of a SALC in the mediastinum, mostly based on mouse data. HEV, high endothelial venule; NKT, natural killer cell; T = T lymphocyte; B1a and B1b, innate B lymphoid cells; B2, conventional B lymphocyte; ILC2, innate (T) lymphoid cell other than NKT cell; APC, antigen-presenting cell. Figure 3. View largeDownload slide Macrophage-rich and lymphocyte-rich SALCs, adult rats. A, Diaphragm with the transition between the broad muscular and the thin tendinous part (arrowhead) and the left retrocardiac pleural fold (arrow) with a few SALCs. B, Detail of a macrophage-rich SALC in the left pleural fold with numerous large macrophages, some lymphocytes, erythrocytes and a few granulocytes. C, A macrophage-rich SALC in the adipose tissue at the hilus of the lungs. D, Two lymphocyte-rich SALCs in the adipose tissue at the hilus of the lungs. E, Detail of 1 of the 2 SALCs from Figure 1D with an adipocyte and several bloodvessels. F, A small lymphocyte-rich SALC in brown adipose tissue on the heart. Hematoxylin & eosin (H&E)-stained sections. Figure 3. View largeDownload slide Macrophage-rich and lymphocyte-rich SALCs, adult rats. A, Diaphragm with the transition between the broad muscular and the thin tendinous part (arrowhead) and the left retrocardiac pleural fold (arrow) with a few SALCs. B, Detail of a macrophage-rich SALC in the left pleural fold with numerous large macrophages, some lymphocytes, erythrocytes and a few granulocytes. C, A macrophage-rich SALC in the adipose tissue at the hilus of the lungs. D, Two lymphocyte-rich SALCs in the adipose tissue at the hilus of the lungs. E, Detail of 1 of the 2 SALCs from Figure 1D with an adipocyte and several bloodvessels. F, A small lymphocyte-rich SALC in brown adipose tissue on the heart. Hematoxylin & eosin (H&E)-stained sections. In the thorax, the majority of lymphoid tissues and organs is located in the mediastinum (Figure 1A). The mediastinal pleura are considered to be the second site in the body with numerous MS/FALCs, after the omentum (mainly rat; Michailova and Usunoff, 2006; Wang et al., 2010), but in mice the pericardium may house even more SALCs (Bénézech et al., 2015). The number and size of SALCs in mediastinal fat can vary between strains of mice and were especially numerous in C57BL/6N (Elewa et al., 2014). SALCs are found also in the retrocardiac pleural folds of rat and mouse (Figure 3A) (Pereira de Sousa et al., 1994). In humans SALCs are located in the mediastinal pleura near the aorta, in the pleura over the esophagus and aorta and in the parietal pleural adipose tissue depots (Aharinejad et al., 1990). Ontogeny In mediastinal fat of mice, numbers of SALCs increase between 3- and 12 months-of-age (Elewa et al., 2014). In humans, pleural SALCs are completely developed at birth with multivacuolated adipocytes, lymphocytes and macrophages, and continue to develop until they reach maximum numbers in infancy (Aharinejad et al., 1990; Michailova and Usunoff, 2006). The numbers then start to decline. Based on omental SALCs in mice, the ontogeny is driven by other molecular events and cells than conventional secondary lymphoid organs, namely by tumor-necrosis factor (TNF) and type 2 innate lymphoid cells, and enhanced by invariant natural killer T cells (Cruz-Migoni and Caamaño, 2016; Perez-Shibayama and Ludewig, 2015). The chemokine Chemokine (C-X-C motif) ligand 13 (CXCL13) (sofar not detected in SALC of rats) is of great importance for the development of SALCs. Like Peyer’s patches, identifiable SALCs can increase in number upon an inflammatory challenge (mouse, omentum), via the recruitment of myeloid cells that express TNF necessary for signaling via the TNF receptors in stromal cells. The distinct postnatal increase in SALCs can lead to the impression that they develop essentially postnatally (Cruz-Mignoni and Caamaño, 2016; Jackson-Jones et al., 2016). Morphology SALCs are often described as aggregates of leukocytes around a central vessel or at a crossroad of vessels, although some authors also distinguish nonvascularized SALCs (Cruz-Migoni and Caamaño, 2016). The microvasculature of SALCs consists of a conventional succession of arteriole, precapillary, capillary, postcapillary, collecting venule, and venule. The capillaries may have dilated portions in the central zone of SALCs. The numbers of HEVs in mediastinal SALCs vary: Elewa et al. (2014) found PNAd+ plump endothelium in mediastinal SALCs of all 3 strains of mice investigated, but especially in the Th1-prone C57BL/6N strain with the largest and most numerous SALCs. There is convincing evidence that most mediastinal SALCs contain efferent lymphatics (lymphatic vessel endothelial receptor 1+ (LYVE-1+); Elewa et al., 2014). Afferent lymphatics appear absent and antigen and particles in the cavities may enter SALCs via the mesothelium, in the same way as (MALT) receives antigen via the specialized follicle-associated epithelium. The SALC-associated mesothelium is plump, and has many microvilli (mouse; mediastinum; Inoue and Otsuki, 1992). General features of SALCs are summarized in Table 1, and Figures 2 and 3. Distinct T and B compartmentalization appears absent. In mediastinal SALC in mice the majority of T cells was found at the base adjacent to adipocytes, while the B cells were located close to the mesothelium (mouse; mediastinum; Inoue and Otsuki, 1992), but other authors do not report this (Cruz-Migoni and Caamaño, 2016; Jaworska-Wilczynska et al., 2016). Iba1+ macrophages and Major histocompatibility complex (MHC) Class II+ antigen-presenting cells were distributed throughout the mediastinal SALCs of mice (Elewa et al., 2014). Germinal centers are absent or at least scarce (mouse; pericardium; Jackson-Jones et al., 2016). Follicular dendritic cells—the scaffold for germinal centers—have not been found in SALCs (mouse, thoracic para-aortic adipose tissue; Newland et al., 2017) and thus germinal center-like formations are probably simpler/more primitive than in MALT and LNs. Macrophage as well as lymphocyte-rich SALCs are present in the mediastinum (Figure 3). Table 1. Characteristics of SALCs, Compared with Conventional Secondary Lymphoid Organs, Tertiary Lymphoid Structures and “Crown-Like” Structures Characteristics  Spleen  LNs  Peyer’s Patches; NALT  Thorax SALCs  TLSa  CLSb  Encapsulation  √  √  —  —  —  —  HEVs  √  √  √  √  √  —  Afferent lymphatics  √  √  —  —  —  —  Efferent lymphatics  √  √  √  √  ?  —  Entrance of antigen or foreign material  Via afferent lymphatics  Via afferent lymphatics  Via follicular-associated epithelium, containing M cells  Via (specialized?) mesothelium  From surrounding, inflamed tissue  NA  T–B compartments  √  √  √  ?  ±  —  Germinal centres  √  √  √  Scarce, possibly simple/primitive follicles and germinal lefts, due to lack of scaffold (follicular dendritic cells)  √  —  Follicular dendritic cells  √  √  √  —  √  —  Innate T lymphoid ILC2 cells  Limited numbers  Scarce?  ?  Significant numbers, especially NHCs  ?  ?  Innate B1 B lymphoid cells  Limited numbers, mainly B1b B cells  —  Scarce?  Large numbers; the presence of B1a B cells in the rat is questionable  ?  ?  Characteristics  Spleen  LNs  Peyer’s Patches; NALT  Thorax SALCs  TLSa  CLSb  Encapsulation  √  √  —  —  —  —  HEVs  √  √  √  √  √  —  Afferent lymphatics  √  √  —  —  —  —  Efferent lymphatics  √  √  √  √  ?  —  Entrance of antigen or foreign material  Via afferent lymphatics  Via afferent lymphatics  Via follicular-associated epithelium, containing M cells  Via (specialized?) mesothelium  From surrounding, inflamed tissue  NA  T–B compartments  √  √  √  ?  ±  —  Germinal centres  √  √  √  Scarce, possibly simple/primitive follicles and germinal lefts, due to lack of scaffold (follicular dendritic cells)  √  —  Follicular dendritic cells  √  √  √  —  √  —  Innate T lymphoid ILC2 cells  Limited numbers  Scarce?  ?  Significant numbers, especially NHCs  ?  ?  Innate B1 B lymphoid cells  Limited numbers, mainly B1b B cells  —  Scarce?  Large numbers; the presence of B1a B cells in the rat is questionable  ?  ?  Abbreviations: NALT, nasopharynx-associated lymphoid tissue; TLS, tertiary lymphoid structures; CLS, Crown-like structures; NA, not applicable. a TLS, lymphocyte-specific microdomains in non-lymphoid organs, formed under chronic inflammatory conditions (Ager, 2017). b CLS, low-grade inflammation present in visceral fat of obese mice and humans with metabolic disease, but—unlike SALCs—also present in subcutaneous fat. Table 1. Characteristics of SALCs, Compared with Conventional Secondary Lymphoid Organs, Tertiary Lymphoid Structures and “Crown-Like” Structures Characteristics  Spleen  LNs  Peyer’s Patches; NALT  Thorax SALCs  TLSa  CLSb  Encapsulation  √  √  —  —  —  —  HEVs  √  √  √  √  √  —  Afferent lymphatics  √  √  —  —  —  —  Efferent lymphatics  √  √  √  √  ?  —  Entrance of antigen or foreign material  Via afferent lymphatics  Via afferent lymphatics  Via follicular-associated epithelium, containing M cells  Via (specialized?) mesothelium  From surrounding, inflamed tissue  NA  T–B compartments  √  √  √  ?  ±  —  Germinal centres  √  √  √  Scarce, possibly simple/primitive follicles and germinal lefts, due to lack of scaffold (follicular dendritic cells)  √  —  Follicular dendritic cells  √  √  √  —  √  —  Innate T lymphoid ILC2 cells  Limited numbers  Scarce?  ?  Significant numbers, especially NHCs  ?  ?  Innate B1 B lymphoid cells  Limited numbers, mainly B1b B cells  —  Scarce?  Large numbers; the presence of B1a B cells in the rat is questionable  ?  ?  Characteristics  Spleen  LNs  Peyer’s Patches; NALT  Thorax SALCs  TLSa  CLSb  Encapsulation  √  √  —  —  —  —  HEVs  √  √  √  √  √  —  Afferent lymphatics  √  √  —  —  —  —  Efferent lymphatics  √  √  √  √  ?  —  Entrance of antigen or foreign material  Via afferent lymphatics  Via afferent lymphatics  Via follicular-associated epithelium, containing M cells  Via (specialized?) mesothelium  From surrounding, inflamed tissue  NA  T–B compartments  √  √  √  ?  ±  —  Germinal centres  √  √  √  Scarce, possibly simple/primitive follicles and germinal lefts, due to lack of scaffold (follicular dendritic cells)  √  —  Follicular dendritic cells  √  √  √  —  √  —  Innate T lymphoid ILC2 cells  Limited numbers  Scarce?  ?  Significant numbers, especially NHCs  ?  ?  Innate B1 B lymphoid cells  Limited numbers, mainly B1b B cells  —  Scarce?  Large numbers; the presence of B1a B cells in the rat is questionable  ?  ?  Abbreviations: NALT, nasopharynx-associated lymphoid tissue; TLS, tertiary lymphoid structures; CLS, Crown-like structures; NA, not applicable. a TLS, lymphocyte-specific microdomains in non-lymphoid organs, formed under chronic inflammatory conditions (Ager, 2017). b CLS, low-grade inflammation present in visceral fat of obese mice and humans with metabolic disease, but—unlike SALCs—also present in subcutaneous fat. The mesothelial lining has an interrupted basal lamina, which facilitates macrophage and lymphocyte migration, and small groups of macrophages are located within the mesothelial lining. Thin, mainly unmyelinated nerve fibers innervate SALCs, in the vicinity of the mesothelial lining, close to the lymphatic vessels and between the cells (mainly rat, pleura, pericardium; Michailova and Usunoff, 2006). Lymphocyte Populations An important feature of SALCs in the abdominal and thoracic cavities is the presence of innate lymphoid cells in addition to the conventional lymphocyte subpopulations (mouse, Cruz-Mignoni and Caamaño, 2016; Elewa et al., 2014, 2016, 2017; Jackson-Jones et al., 2018; Koyasu and Moro, 2012; Saenz et al., 2010). SALCs contain innate lymphocyte type 2 (ILC2) cells (especially natural helper cells or NHCs), which can secrete type 2 cytokines (eg, interleukin-5, interleukin-13). About half of the B lymphocytes present in the pleural spaces are nonconventional, innate B lymphoid cell (B1) cells (sIgMhi, sIgDlow, CD11b+), which are nonconventional innate B lymphocytes (as opposed to the conventional Conventional B lymphocyte [B2] lymphocytes). B1 B cells are also present in the spleen and intestines, but they are CD11b−. CD5+ B1a cells, a subpopulation of B1 cells, are not well-documented in the rat and may be scarce (Berland and Wortis, 2002). Box 1 compares the properties of CD5+ innate B lymphoid cell (B1a), CD5– innate B lymphoid cell (B1b), and B2 cells, and their presence in rat, mouse, and man. Box 1. Comparison of B1 (Innate) and B2 (Conventional) Lymphocytes B1a and B1b cells develop early in the fetal liver and are self-renewing in situ; B2 cells develop late in the bone marrow and are replaced from the bone marrow. B1a and B1b cells are primarily located in the peritoneal and pleural cavities; B2 cells in the spleen, LNs and MALT. B1a cells have a high production of natural antibodies; production by B1b cells is poorly investigated; production by B2 cells is low. Antibody isotype of B1a and B1b cells is IgM and antibody avidity is low; isotypes of B2 are IgM, IgG, IgD, and IgA and their avidity is high B1a antigens are often carbohydrate-specific and rarely protein-specific; B1b antigens are possibly carbohydrate- and protein-specific; B2 antigens are protein-specific and rarely carbohydrate-specific. B1a memory development is little or nonexistent; B1b memory (IgM) and B2 memory is present. B1a responses are T cell-independent; B1b responses are largely T cell-independent; B2 responses are often T cell-dependent. Evidence for the presence of B1a cells is sufficient in mouse and man, but poor in rat; evidence for B1b cells is sufficient in mouse and rat, but limited in man; evidence for B2 cells is sufficient in mouse, rat and man (Cunningham et al., 2014; Perry et al., 2012; Popi et al., 2016) Function SALCs are secondary lymphoid organs, involved in the induction of adaptive immune responses, with antigen-presenting cells in the context of MHC II complexes and antigen-specific B cells undergoing expansion and Ig switching even in mice lacking spleen, lymph nodes (LNs), and Peyer’s patches (mouse; mediastinum and omentum; Jackson-Jones et al. 2016; Rangel-Moreno et al. 2009). However, Meza-Perez and Randall (2017; mouse; omentum) positioned them between secondary and tertiary lymphoid structures. SALCs are key in serous B cell homeostasis and activation, by retaining B1 cells in the serous cavities via high expression of the chemokine CXCL13 (mouse; Rangel-Moreno et al., 2009). The presence of B1 cells and ILC2 T cells strongly suggest that SALCs support and coordinate the activation of innate lymphocytes in early, acute immune responses (mouse; peritoneum; Bénézech et al., 2015). IMMUNE FUNCTIONS OF THE MESOTHELIUM AND VISCERAL ADIPOSE TISSUE Mesothelium The mesothelial cells are mostly flattened, but some are cuboidal or “cobble stone”-like (Jaworska-Wilczynska et al., 2016). The cuboidal mesothelial cells are often found lining SALCs; they have abundant microvilli and occasionally cilia and their cytoplasm is rich in organelles, including multilamellar bodies (Mutsaers et al., 2002). The multilamellar bodies resemble those of pneumocytes II, which produce lung surfactant. The mesothelium has many functions to maintain serosal homeostasis and repair (rat and mouse; pleura and omentum; Gupta and Gupta, 2015; Katz et al., 2011; Mutsaers et al., 2002). It is a player in innate and acquired immune mechanisms, namely as a nonprofessional antigen-presenting cell (expressing MHC class II) and as producer of cytokines, leukocyte chemoattractants and glycosaminoglycans. Adipose Tissue Apart from thermogenesis and energy storage and release, visceral adipocytes and their stromovasculature play a pivotal role in immunity (acquired immunity: Kaminski and Randall, 2010; innate immunity: Schaffler and Scholmerich, 2010). Most information is based on investigations of the white adipose tissue in the abdominal cavity, but apply also to the mixed white and brown adipose tissue in the thorax (Baragetti et al., 2016; Patil et al., 2014). The white adipocytes produce and secrete classical cytokines such as interleukin-6 and TNF, and hormones like leptin. Leptin regulates macrophage function and in a broader sense modulates the immune functions of adipocytes by eg, inducing Toll-like receptor expression (Schaffler and Scholmerich, 2010). Adipocytes express multiple Toll-like receptors to recognize pathogens and to initiate immune responses, and they can secrete a variety of monocyte/macrophage chemoattractant molecules (Schaffler and Scholmerich, 2010; Dalmas et al., 2011). Interestingly, the innate immune system itself influences beige fat formation, via type 2 cytokine signaling (Kissig et al., 2016). For example, interleukin-4 production in white adipose tissue stimulates macrophages to produce norepinephrine, which leads to beige fat activation. The immune system-related aspects of the adipose tissue may depend on the location (Pond, 2005): adipocytes around LNs and omental SALCs are relatively small compared with those further away, respond more strongly to cytokines, and neighbor numerous antigen-presenting dendritic cells. Moreover, adipose tissue around the inguinal LN of mice contained less gamma/delta T lymphocytes and NK cells than epidydimal fat (Caspar-Bauguil et al., 2005). In the rat, perivascular adipose tissue in the abdomen express more inflammatory genes and markers of immune cells than the perivascular fat in the thorax, possibly because the abdominal adipose tissue has white adipose tissue characteristics, whereas in the thorax it is beige (Padilla et al., 2013). SALCs AND THORACIC FLUID RESPONSES TO EXPOSURE AND IN DISEASE The responses of mediastinal SALCs and thoracic fluid to inflammogenic stimuli, mostly inhaled or instilled particles, are summarized in Box 2 (rat and mouse; Bernstein et al., 2015; Broaddus et al., 2011; McKenzie and Caamaño, 2015; Mercer et al., 2013; Murphy et al., 2011; Ryman-Rasmussen et al., 2009; Gelzleichter et al., 1999; Choe et al., 1997; Peao et al., 1992; Bénézech et al., 2015; Jackson-Jones et al., 2016; Cooray, 1949; Pereira de Sousa et al., 1994). Acceleration of fluid flow happens by widening of already opened stomata and opening of formerly closed stomata, by increased drainage of the pleural interstitium and by increased diffusion and transcytosis (Bodega and Agostoni, 2004; Li and Li, 2004). Box 1. Exposure-Induced Changes in Mesothelium, Thoracic Fluid and Mediastinal SALCs (MS and FALCs) of Rodents Flat mesothelial cells become cuboidal and release inflammatory mediators (mouse; thoracic serosa); mesothelial cells may proliferate (rat, mouse; pleura). The fluid flow from the cavities into the lymphatics accelerates (rat; pleural space), and harbor increased numbers of inflammatory cells (rat; pleural space). Macrophages are recruited and activated, comparable to alveolar macrophages (rat; pleura, pleural space). Local serosal inflammation may occur (rat, mouse; pleura). SALCs increase in size and cellularity, and may fuse (rat, mouse; mediastinum); they may also develop germinal center-like B2 cells (GL7 + Ki67+) and germinal center-like structures (mouse; mediastinum). Particles can accumulate in SALCs and may induce granulomata and cell death in SALCs (rat; mediastinum). In humans, activation of SALCs has been linked also to inhalation of particles (Boutin et al., 1996; Mitchev et al., 2002). Black spots on the parietal pleura can be anthracotic SALCs, whereas hyaline pleural plaques, associated with exposure to asbestos, may be anthracotic SALCs or inflammatory processes on the parietal pleura as a result of blockage of the stomata by fibers (Murphy et al., 2011). Based on the stimulus, either mainly thoracic or abdominal SALCs react. For example omental SALCs reacted much more than pleural SALCs upon percutaneous injection with Schistosoma mansonii in mice, a nematode with preference to the mesenteric veins (Panasco et al., 2010). The opposite happened upon a subcutaneous injection with Litomosoides sigmodontis, a nematode that resides in the pleural cavity (Jackson-Jones et al., 2016). Particles and particle-containing phagocytes are thought to be removed from the pleural space via the stomata and associated lymphatic channels, but Lehnert (1992) questioned this route as the only or most important pathway. Based mainly on rat and some mouse and human data, he hypothesized that the caudal mediastinal tissue acts as the primary site via which particle-containing phagocytes are removed from the pleural space, to end up in the local LNs. Diseases can also affect SALCs. The mediastinal SALCs in the autoimmune MRL/MpJ-lpr mouse (prone to a systemic lupus erythematosus-like disease) and the BXSB/MpJ-Yaa mouse (prone to systemic autoimmunity) were much larger, with high numbers of T and B cells, macrophages and proliferating cells than their respective controls (Elewa et al., 2016). The size of SALCs correlated well with immune cell infiltration in the lungs. During pleural and lung inflammation, pleural B1 cells migrate to SALCs of the mediastinum and pericardium and produce IgM in the thoracic cavities (Jackson-Jones and Benezech, 2018). Moreover, B1 cells in peri-aortic SALCs appear to protect against (local) atherosclerosis (Srikakulapu et al., 2017). Pleural fluid cell numbers are increased in humans and mice with systemic autoimmune diseases (Pfau et al., 2014). SALCs may play a role in cancer metastasis as well, as shown in the peritoneum: some cancer cells expressed the receptors chemokine (C-C) receptor 4 and chemokine (C-X-C) receptor 4, which can favor their migration to SALCs, because their cell populations can express their ligands (Chemokine (C-C) motif 22[CCL22] and CXCL12) (Cruz-Migoni and Caamaño, 2016). DISCUSSION AND FUTURE DIRECTIONS The serosal immune system has unique anatomic and morphologic features, namely the lymphoid structures (SALCs), the large quantity of innate B1 B and ILC2 lymphoid cells and the lymphatic drainage units. The immune physiology of the serosal cavities is still poorly understood and many questions still remain. Do the abdominal and thoracic cavities co-operate like the respiratory tract and gut in the mucosal immune system? The different cavities start as 1 celom in embryonic life, but after birth there is no bulk flow between the pleural and peritoneal cavities (Grimaldi et al., 2006). Still, ip-injected material in the rat is rapidly transported through lymphatics to the LNs in the mediastinum (Shibata et al., 2007). This suggests a co-operation between the cavities on both sides of the diaphragm, albeit possibly only under “overload” conditions. Moreover, experiments with nematode-infected mice suggest that the cavities act largely as independent environments (Panasco et al., 2010). Another question is whether the mesothelial epithelium lining the SALCs can take up antigens and act in a way comparable to the follicle-associated epithelium of Peyer’s patches and nasopharynx-associated lymphoid tissue? Interestingly in animals with an undivided celom, like for example insects, the serosal epithelium is immune-competent and expresses many genes involved in bacterial recognition and transduction of this recognition to receptor activation (Jacobs et al., 2014). As such, it is especially involved in acute and mainly innate immune responses. B1 cells in the cavities not only protect local tissues, but also the mucosae with an acute innate immune response (Cruz-Migoni and Caamaño, 2016; Jackson-Jones et al., 2016; Symowski and Voehringer, 2017). 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Toxicological SciencesOxford University Press

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